Project Summary X-ray fluorescence microscopy (XFM) has emerged as powerful tool to visualize metals within cells or tissue. Using X-ray microprobes, investigators can now produce elemental maps of biological material at the cellular and subcellular levels. Determining the elemental distribution and concentration at such resolution, however, is of little use without the option to also identify subcellular compartments or localizing key proteins with which the element in question is associated. To date, without additional, correlative techniques, it is impossible to do so unless the organelle in question shows an increased concentration in one of the detected elements (e.g. phosphorus to identify the nucleus). This proposal seeks to develop a novel robust technique in which elemental imaging and organelle localization is done simultaneously, as part of the regular XFM scan and at energies ?10 keV. In Specific Aim1 we will synthesize nanoparticle containing probes that are tethered to antibodies via click chemistry. The nanoparticles will either contain Ni and/or Co as the central metal and the antibodies will be against organelle markers such as Lamp1 for lysosomes. Using XFM we will then be able to visualize the organelles, albeit indirectly, through their Ni (or Co) signature and the element of interest in one scan. The application of Specific Aim1 is limited to chemically fixed samples. In other experiments, direct, in vivo labeling might be desired to avoid elemental redistribution due to chemical fixation and permeabilization processes. This is especially important when the elements of interests are only loosely bound. In Specific Aim 2 we will therefore design and synthesize hybrid lipid-coated nickel nanoparticles of which the surface architecture will be tuned to target specific organelles.